CN113584092A - Method for enriching EPA and DHA in fish oil through enzymatic hydrolysis - Google Patents
Method for enriching EPA and DHA in fish oil through enzymatic hydrolysis Download PDFInfo
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Abstract
The invention belongs to the technical field of enriching specific fatty acid in grease, and discloses a method for enriching EPA and DHA in fish oil by enzymatic hydrolysis, which comprises the following steps: (1) mixing fish oil, water and lipase, and performing hydrolysis reaction at 20-70 deg.C until the acid value of the hydrolysate is 60 + -15 mg/g; (2) centrifuging the hydrolysate obtained in the step (1), taking the upper oil phase, mixing the upper oil phase with water and partial glyceride lipase to continue hydrolysis reaction, and stopping the reaction when the acid value of the hydrolysate is 70 +/-15 mg/g; (3) and (3) centrifuging the hydrolysate in the step (2), taking the upper oil phase, and performing molecular distillation to obtain a heavy phase which is mixed glyceride rich in EPA and DHA. The hydrolysis method directly uses the fish oil and the water as raw materials, is environment-friendly, green and safe, is simple to operate, does not consume reagents, and reduces the cost, and the DHA and the EPA can account for 45.53-60.45 percent of the fish oil after two steps of hydrolysis.
Description
Technical Field
The invention belongs to the technical field of enrichment of specific fatty acids in grease, and particularly relates to a method for enriching EPA and DHA in fish oil through enzymatic hydrolysis.
Background
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) belong to the omega-3 polyunsaturated fatty acid series, which are also the major components of the cell membranes of brain and retinal tissues.
However, EPA and DHA must be taken through food as essential fatty acids that the human body cannot synthesize by itself. At present, the supplement of human EPA and DHA is mainly derived from commercial fish oil, but the content of EPA and DHA in natural fish oil glyceride is generally low (< 25%), and the supplement can not meet the requirement of human body in the fields of medicine and health care products. Therefore, the natural fish oil which is sold in the market and is rich in EPA and DHA to meet the needs of human bodies has great market prospect. The DHA and EPA products mainly have the enrichment forms of free type, ethyl ester type and glyceride type, wherein the free type EPA and DHA are difficult to accept by people due to taste acid and are extremely easy to oxidize, decay and deteriorate to form substances harmful to human bodies; the ethyl ester type EPA and DHA are difficult to absorb and digest by human bodies when eaten, and potential safety hazards exist; therefore, only the glyceride type EPA and DHA can meet the absorption and digestion requirements of the human body, and the glyceride type EPA, DHA and triglyceride type EPA, DHA are included. Diglyceride is a structural lipid formed by replacing one fatty acid of Triglyceride (TAG) by hydroxyl, consists of two structural isomers, 1,2-DAG and 1,3-DAG, and is a part of the natural composition of edible oil. Diglycerides are classified as safe food (GRAS) by the U.S. Food and Drug Administration (FDA) and have wide application in the food field.
The current methods for enriching EPA and DHA are divided into traditional methods and enzymatic methods. The conventional method comprises the following steps: low temperature crystallization, urea inclusion, molecular distillation, silver ion complexation, and supercritical fluid extraction. The traditional method has the defects of high cost, complex process, large amount of solid wastes, long reaction time, low reaction yield and the like in the process of enriching EPA and DHA, and simultaneously has the oxidation loss of EPA and DHA in the enriching process. The industrial enrichment of EPA and DHA mostly adopts enzyme enrichment, wherein triglyceride type EPA and DHA are mainly synthesized by an enzyme method for enrichment. The method for synthesizing triglyceride by utilizing enzymatic catalysis and enrichment of EPA and DHA mainly comprises three catalytic forms of esterification, ester exchange and hydrolysis, but the EPA and DHA have long carbon chains and homeopathic double bonds, so that the EPA and DHA have large spatial structures, and therefore, the yield is low and the reaction time is long when esterification and ester exchange are carried out.
In Lijun's Shuichi thesis research on preparation of high-content polyunsaturated fatty acid fish oil by Aspergillus oryzae lipase, domestic Engraulis japonicus oil is used as a raw material, and a high-content EPA and DHA glyceride product is prepared by two-step enrichment of enzymatic alcoholysis and transesterification, wherein the reaction conditions are optimized, and after 48 hours of reaction, the total content of EPA and DHA in the glyceride product can reach 41.78%. Li LiSai et al published "DHA and EPA glycerides enriched by immobilized lipase selective hydrolysis of waste fish oil" in "bioprocessing Process" 2009, volume 7, phase 6. the text adopts two hydrolysis steps, the first hydrolysis step is followed by the second hydrolysis step, and the EPA and DHA mass fractions are respectively increased from 4.2% and 18.9% of the original fish oil to 8.5% and 42% by the two hydrolysis steps. In Liufang, in Shuo thesis research on triglyceride rich in EPA and DHA through enzymatic catalysis, refined deep sea fish oil is used as a raw material, firstly, lipase Amano AY 'Amano' 400SD is used as a catalyst to hydrolyze the raw material, then, glyceride obtained through hydrolysis reaction and concentrated fish oil ethyl ester are used for synthesizing triglyceride through enzymatic transesterification reaction under vacuum condition, and the content of EPA and DHA in the product glyceride is respectively 25.3 wt% and 21.6 wt%. The methods enrich EPA and DHA to a certain extent, but compared with the two-step hydrolysis method of the invention, the methods have the defects of long reaction time, high energy consumption, low enrichment rate and the like, and the products obtained by the enrichment of the methods contain a certain amount of monoglyceride and free fatty acid, thus affecting the product quality and being incapable of being used as a method for industrially enriching EPA and DHA.
Disclosure of Invention
The invention aims to make up the defects of the existing enrichment technology and EPA and DHA product types, and provides a method for enriching EPA and DHA in fish oil by enzymatic hydrolysis.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for enriching EPA and DHA in fish oil by enzymatic hydrolysis comprises the following steps:
(1) mixing fish oil, water and lipase, stirring, performing hydrolysis reaction at 20-70 deg.C, and stopping reaction when acid value of hydrolysate is 60 + -15 mg/g;
(2) centrifuging the hydrolysate obtained in the step (1), taking the upper oil phase, mixing with water and partial glyceride lipase, stirring, and continuing the hydrolysis reaction at 20-70 ℃, and stopping the reaction when the acid value of the hydrolysate is 70 +/-15 mg/g;
(3) and (3) centrifuging the hydrolysate obtained in the step (2), taking the upper oil phase, and performing molecular distillation to obtain a heavy phase, namely the mixed glyceride rich in EPA and DHA.
Preferably, the lipase of step (1): water: the mass ratio of the fish oil is (0.0001-0.0075): (0.05-0.5): 1.
preferably, the partial glyceride lipase of step (2): water: the mass ratio of the upper oil phase is (0.005-0.075): (0.05-0.5): 1.
preferably, when the acid value of the hydrolysate in the step (1) is 60 +/-5 mg/g, stopping the reaction; and (3) stopping the reaction when the acid value of the hydrolysate in the step (2) is 70 +/-5 mg/g.
Preferably, the hydrolysis reaction temperature in the step (1) is 35-45 ℃, and the hydrolysis reaction time is 1-6 h; and (3) the hydrolysis reaction temperature in the step (2) is 35-45 ℃, and the hydrolysis reaction time is 1-6 h.
Preferably, the Lipase in the step (1) is one or a mixture of two of Lipase TL100L and Lipase T1.
Preferably, the partial glyceride Lipase in the step (2) is one or a mixture of more than two of Lipase AOL, Lipase AOL-V269D and Lipase SMG 1.
Preferably, the pH of the hydrolysis reaction in the step (1) and the step (2) is 6-8, and the hydrolysis reaction is carried out under the protection of inert gas.
Preferably, the molecular distillation treatment in the step (3) is carried out under the conditions of a vacuum degree of 0.01-0.2 pa, a transmembrane rotation speed of 250-350 rpm, a feeding speed of 1.0-1.5 ml/min and a distillation temperature of 120-160 ℃.
Preferably, the stirring rate of the hydrolysis reaction in the step (1) is 200-1000 rpm; the stirring speed of the hydrolysis reaction in the step (2) is 200-1000 rpm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the traditional physical and chemical enrichment method is not involved in the process of enriching EPA and DHA, and reagents are not consumed, so that the cost is reduced. Meanwhile, the hydrolysis method directly uses fish oil and water as raw materials, and has the advantages of environmental friendliness, greenness, safety and simplicity in operation.
(2) The method creatively uses two lipases to hydrolyze the fish oil, firstly, the Lipase Lipase TL100L is utilized to hydrolyze the fish oil, when the acid value is measured to be in the range of 60 +/-5 mg/g, the partial glyceride Lipase AOL-V269D is further utilized to selectively hydrolyze the saturated fatty acid and the monounsaturated fatty acid in the diglyceride and the monoglyceride in the mixed glyceride after centrifugal dehydration and enzyme removal, thereby realizing the further purification of EPA and DHA in the mixed glyceride.
(3) The product obtained by the method is rich in diglyceride DHA and EPA, and diglyceride can reduce excessive accumulation of human body fat and promote lipid metabolism in human body as a functional oil, so that the product obtained by the method has wide application in the fields of food and medicine.
(4) After two-step hydrolysis, the content of DHA and EPA in the fish oil is 45.53-60.45%, which is 18.14-27.69% higher than the content of EPA and DHA in the original fish oil; after two-step hydrolysis, the EPA and the DHA account for 60.45 percent of tuna oil in the maximum ratio, which is 9.95 percent and 13.55 percent respectively higher than the total content of the EPA and the DHA in glyceride products of Lilisfan (50.5 percent) and Liufang (46.9 percent) in the existing technology; after two-step hydrolysis, the highest ratio of EPA and DHA in algae oil is 59.68% in total, which is 21.88% higher than that of the prior art that the DHA is enriched by the enzyme hydrolysis of once sweet (the highest ratio is 37.8%).
Drawings
FIG. 1 is a diagram of the second hydrolysis reaction.
FIG. 2 is a graph showing the effect of the second hydrolysis reaction after centrifugation.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Lipase TL100L, a Lipase used in the examples, was purchased from Denmark Novoxin;
partial glyceride Lipase Lipase AOL and Lipase AOL-V269D are disclosed in patent CN108504643A, "partial glyceride Lipase from Aspergillus oryzae, its preparation method and crystal structure".
Tuna oil, salmon oil, anchovy oil, and winterized algae oil were purchased from fengyi lipidemics (shanghai) limited.
Example 1
Adding 500g of tuna oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system to be 40 ℃, and stirring at the speed of 200 rpm; wherein the addition amount of the water is 15% of the mass of the tuna oil, and the addition amount of the Lipase TL100L is 0.1% of the mass of the tuna oil.
After 3.5h of reaction, the acid value of the hydrolyzate was found to be 60.36mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 20% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 2.0% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 1.5h, an acid value of 68.26mg/g of the hydrolyzate was detected, at which point the hydrolysis was terminated by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 1.
Table 1: composition and content of tuna oil crude oil two-step hydrolysate
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 140 ℃; 244.7g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment is collected, and 126.5g of light phase (mainly fatty acid) is obtained; the yield of the heavy phase obtained from the preparation of example 1 was 48.89% (note: yield-mass of heavy phase/mass of said tuna oil crude).
The components of the crude tuna oil and the heavy-phase oil prepared by molecular distillation in example 1 were identified by GC-FID analysis, and the identification results are shown in table 2. The results show that the EPA and DHA content in tuna oil after two-step hydrolysis is 56.45% in total, which is 27.69% higher than the EPA and DHA content in tuna crude oil (28.76%), and that in the existing technology, Lijun is enriched to prepare a glyceride product with high EPA and DHA content through two-step enzymatic alcoholysis and ester exchange reaction, the total content of EPA and DHA in the glyceride product is only 39.72%, and the content in this example is 16.73% higher than that in this example.
Table 2: tuna oil crude oil and the product obtained by molecular distillation contain the main fatty acid content
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Example 2
Adding 500g of salmon oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 10% of the quality of the salmon oil, and the addition amount of the Lipase TL100L is 0.15% of the quality of the salmon oil.
After 3.5h of reaction, the acid value of the hydrolyzate was found to be 58.37mg/g, at which point stirring was stopped to terminate the hydrolysis reaction. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 20% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 2.5% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 1.5h, the acid value of the hydrolyzate was found to be 70.55mg/g, at which point the hydrolysis reaction was terminated by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 3.
Table 3: component composition and content of two-step hydrolysate of crude salmon oil
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 140 ℃; 228.3g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment is collected, and 128.6g of light phase (mainly fatty acid) is obtained; the yield of the heavy phase obtained by the preparation of example 2 was 45.66% (note: yield-mass of heavy phase/min)The quality of the salmon oil crude oil).
The components of the salmon crude oil and the heavy-phase oil prepared by molecular distillation in example 2 were identified by GC-FID analysis, and the identification results are shown in table 4. The results show that the EPA and DHA accounts for 49.11% in the salmon oil in the highest ratio after hydrolysis, and the ratio of the EPA and the DHA in the salmon oil (25.08%) is improved by 24.03%.
Table 4: content of main fatty acid in crude oil of salmon oil and product obtained by molecular distillation
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Example 3
Adding 500g of salmon oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system at 45 ℃ and the stirring speed at 200 rpm; wherein the addition amount of the water is 20% of the quality of the salmon oil, and the addition amount of the Lipase TL100L is 0.1% of the quality of the salmon oil.
After 2.0h of reaction, the acid value of the hydrolyzate was found to be 58.37mg/g, at which point stirring was stopped to terminate the hydrolysis reaction. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 45 ℃ and the stirring speed at 200 rpm; wherein the addition amount of the water is 15% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 1.5% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 2.0h, the acid value of the hydrolyzate was found to be 67.36mg/g, at which point the hydrolysis was stopped by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 5.
Table 5: component composition and content of two-step hydrolysate of crude salmon oil
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0mL/min, and distillation temperature of 130 ℃; collecting 214.3g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment, and 144.8g of light phase (mainly fatty acid); the yield of the heavy phase obtained from the preparation of example 3 was 42.86% (note: yield-mass of heavy phase/mass of crude salmon oil).
The components of the salmon crude oil and the heavy-phase grease prepared by molecular distillation in example 3 were identified by GC-FID analysis, and the identification results are shown in table 6, which indicates that the highest ratio of EPA and DHA in the salmon oil after hydrolysis totals 46.8%, which is 21.72% higher than the ratio of EPA and DHA in the salmon crude oil (25.08%).
Table 6: content of main fatty acid in crude oil of salmon oil and product obtained by molecular distillation
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Example 4
Adding 500g of anchovy tail oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 15% of the weight of the anchovy oil, and the addition amount of the Lipase TL100L is 0.1% of the weight of the anchovy oil.
After 3.5h of reaction, the acid value of the hydrolyzate was found to be 62.38mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 25% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 2.0% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 1.5h, the acid value of the hydrolyzate was found to be 74.32mg/g, at which point the hydrolysis reaction was terminated by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 7.
Table 7: the two-step hydrolysate of crude oil of Pteris multifida oil has composition and content
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 140 ℃; 255.4g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment is collected, and 118.7g of light phase (mainly fatty acid) is obtained; the yield of the heavy phase obtained from the preparation of example 4 was 51.08% (note: yield-mass of heavy phase/mass of the anchovy oil crude).
The components of the anchovy crude oil and the heavy-phase oil prepared by molecular distillation in example 4 were identified by GC-FID analysis, and the identification results are shown in table 8. The result shows that the EPA and the DHA account for 45.53% in the anchovy oil in the maximum ratio after hydrolysis, and the EPA and the DHA account for 24.48% in the anchovy crude oil (21.05%).
Table 8: the main fatty acid content in the crude oil of Pteris multifida oil and the product obtained by molecular distillation
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Example 5
Adding 500g of tuna oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system to be 45 ℃ and the stirring speed to be 200 rpm; wherein the addition amount of the water is 20% of the mass of the tuna oil, and the addition amount of the Lipase TL100L is 0.2% of the mass of the tuna oil.
After 1.5h of reaction, the acid value of the hydrolyzate was found to be 57.97mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 45 ℃ and the stirring speed at 200 rpm; wherein the addition amount of the water is 20% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 1.5% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 1.5h, the acid value of the hydrolyzate was found to be 68.82mg/g, at which point the hydrolysis was stopped by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 9.
Table 9: composition and content of tuna oil crude oil two-step hydrolysate
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 160 ℃; collecting 207.6g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment, and 142.8g of light phase (mainly fatty acid); the yield of the heavy phase obtained from the preparation of example 5 was 41.52% (note: yield-mass of heavy phase/mass of said tuna oil crude).
The components of the crude tuna oil and the heavy-phase oil prepared by molecular distillation in example 5 were identified by GC-FID analysis, and the identification results are shown in table 10. The results show that the EPA and DHA content in tuna oil after hydrolysis is 60.45% in total, which is 18.14% higher than the EPA and DHA content in tuna crude oil (28.76%). In the existing technology, plum lisfan is enriched to prepare a glyceride product with high content of EPA and DHA by a two-step hydrolysis method, the total content of EPA and DHA in the glyceride product is 50.5% at most, Liu Fang is hydrolyzed by an enzyme method to prepare a glyceride product with high content of EPA and DHA, the total content of EPA and DHA in the glyceride product is 46.9% at most, and the content of EPA and DHA in the glyceride product is respectively improved by 9.95% and 13.55% compared with that of plum lisfan and Liu Fang.
Table 10: tuna oil crude oil and the product obtained by molecular distillation contain the main fatty acid content
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Example 6
Since the fatty acid of the algae oil is similar to the fish oil and is rich in EPA and DHA, the invention is also suitable for extracting the mixed glyceride rich in EPA and DHA from the algae oil. Adding 500g of winterized algae oil, distilled water and Lipase TL100L into a reaction container, fully stirring to completely mix substances in the reaction container, introducing nitrogen to protect reaction products, sealing the reaction container, controlling the temperature of a reaction system at 40 ℃, and controlling the stirring speed at 200 rpm; wherein the addition amount of the water is 20% of the mass of the winterized algae oil, and the addition amount of the Lipase TL100L is 0.1% of the mass of the winterized algae oil.
After 6h of reaction, the acid value of the hydrolyzate was 55.23mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of water is 25% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 2.0% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 2.5h, an acid value of 66.32mg/g of the hydrolyzate was detected, at which point the hydrolysis was terminated by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 11.
Table 11: components and contents of two-step hydrolysate of winterized algae oil crude oil
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 140 ℃; 266.5g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment is collected, and 118.4g of light phase (mainly fatty acid) is obtained; the yield of the heavy phase obtained from the preparation of example 6 was 53.30% (note: yield-mass of heavy phase/mass of winterized algae oil crude oil).
Table 12: main fatty acid content in winterized algae oil crude oil and products obtained by molecular distillation
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
The components of winterized algal oil crude oil and heavy phase oil prepared by molecular distillation in example 6 were identified by GC-FID analysis, and the identification results are shown in table 12. The results show that the highest ratio of EPA and DHA in algae oil after hydrolysis is 59.68%, the ratio of EPA and DHA in the algae oil is increased by 19.72% compared with the ratio of EPA and DHA in winterized algae crude oil (39.96%), the highest ratio of DHA in the once sweet algae in the prior art is only 37.8% after the once sweet algae is enriched by enzymatic hydrolysis, and the ratio of DHA in the once sweet algae is increased by 21.88% in the embodiment.
Comparative example 1
Adding 500g of tuna oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system to be 40 ℃, and stirring at the speed of 200 rpm; wherein the addition amount of the water is 15% of the mass of the tuna oil, and the addition amount of the Lipase TL100L is 1.5% of the mass of the tuna oil.
After 1.0h of reaction, the acid value of the hydrolyzate was found to be 78.26mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. Centrifuging the hydrolysate at 10000rpm for 5min, taking an upper oil phase, adding distilled water and partial glyceride Lipase AOL-V269D to continue hydrolysis reaction, introducing nitrogen to protect the reaction product, sealing the reaction container, controlling the temperature of the reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 20% of the mass of the oil phase collected by centrifugation, and the addition amount of the partial glyceride Lipase AOL-V269D is 2.0% of the mass of the oil phase collected by centrifugation.
After a further reaction time of 1.5h, the acid value of the hydrolyzate was found to be 106.78mg/g, at which point the hydrolysis reaction was terminated by stopping the stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 13.
Table 13: composition and content of tuna oil crude oil two-step hydrolysate
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 140 ℃; 179.8g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment and 258.6g of light phase (mainly fatty acid) obtained by molecular distillation treatment are collected; the yield of the heavy phase obtained for the preparation of comparative example 1 was 35.96% (note: yield-mass of heavy phase/mass of the tuna oil crude).
The components of the crude tuna oil and the heavy-phase oil prepared by molecular distillation in comparative example 1 were identified by GC-FID analysis, and the identification results are shown in table 14. The results show that the highest ratio of EPA and DHA in tuna oil after hydrolysis is only 34.09% in total, and the analysis reason is that the hydrolysis process is rapid and the reaction is difficult to control due to the excessively high enzyme addition amount.
Table 14: tuna oil crude oil and the product obtained by molecular distillation contain the main fatty acid content
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
Comparative example 2
Adding tuna oil, distilled water and Lipase TL100L into a reaction vessel, fully stirring to completely mix substances in the reaction vessel, introducing nitrogen to protect reaction products, sealing the reaction vessel, controlling the temperature of a reaction system at 40 ℃, and stirring at 200 rpm; wherein the addition amount of the water is 15% of the mass of the tuna oil, and the addition amount of the Lipase TL100L is 0.15% of the mass of the tuna oil.
After 2.0h of reaction, the acid value of the hydrolyzate was found to be 61.22mg/g, at which point the hydrolysis reaction was terminated by stopping stirring. The glyceride composition of the hydrolysate was determined by HPLC as shown in Table 15.
Table 15: composition and content of tuna oil crude oil two-step hydrolysate
Centrifuging the hydrolysate at 10000rpm for 5min, and performing molecular distillation treatment on the upper oil phase under the condition of vacuum degree of 10-3mbar, transmembrane rotation speed of 300rpm, feeding rate of 1.0ml/min and distillation temperature of 150 ℃; 288.6g of heavy phase (namely mixed glyceride rich in DHA and EPA) obtained by molecular distillation treatment is collected, and 133.2g of light phase (mainly fatty acid) is obtained; the yield of the heavy phase obtained for the preparation of comparative example 2 was 57.72% (note: yield-mass of heavy phase/mass of the tuna oil crude).
The components of the crude tuna oil and the heavy-phase oil prepared by molecular distillation in comparative example 2 were identified by GC-FID analysis, and the identification results are shown in table 16. The results show that the highest proportion of EPA and DHA in tuna oil is only 33.21% by a single hydrolysis step, which is far less effective than a two hydrolysis step.
Table 16: tuna oil crude oil and the product obtained by molecular distillation contain the main fatty acid content
The obtained heavy phase: centrifuging the hydrolysate, subjecting the upper oil phase to molecular distillation, and collecting the heavy phase
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for enriching EPA and DHA in fish oil by enzymatic hydrolysis is characterized by comprising the following steps:
(1) mixing fish oil, water and lipase, stirring, performing hydrolysis reaction at 20-70 deg.C, and stopping reaction when acid value of hydrolysate is 60 + -15 mg/g;
(2) centrifuging the hydrolysate obtained in the step (1), taking the upper oil phase, mixing with water and partial glyceride lipase, stirring, and continuing the hydrolysis reaction at 20-70 ℃, and stopping the reaction when the acid value of the hydrolysate is 70 +/-15 mg/g;
(3) and (3) centrifuging the hydrolysate obtained in the step (2), taking the upper oil phase, and performing molecular distillation to obtain a heavy phase, namely the mixed glyceride rich in EPA and DHA.
2. The method according to claim 1, wherein the lipase of step (1): water: the mass ratio of the fish oil is (0.0001-0.0075): (0.05-0.5): 1.
3. the method according to claim 1, wherein the partial glyceride lipase of step (2): water: the mass ratio of the upper oil phase is (0.005-0.075): (0.05-0.5): 1.
4. the method according to any one of claims 1 to 3, wherein the reaction is stopped when the acid value of the hydrolysate in the step (1) is 60 ± 5 mg/g; and (3) stopping the reaction when the acid value of the hydrolysate in the step (2) is 70 +/-5 mg/g.
5. The method according to claim 4, wherein the hydrolysis reaction temperature in the step (1) is 35-45 ℃, and the hydrolysis reaction time is 1-6 h; and (3) the hydrolysis reaction temperature in the step (2) is 35-45 ℃, and the hydrolysis reaction time is 1-6 h.
6. The method of claim 1, wherein the Lipase in step (1) is one or a mixture of two of Lipase TL100L and Lipase T1.
7. The method of claim 1, wherein the partial glyceride Lipase of step (2) is one or a mixture of two or more of Lipase AOL, Lipase AOL-V269D and Lipase SMG 1.
8. The method according to claim 1, wherein the hydrolysis reaction of step (1) and step (2) has a pH of 6-8, and is carried out under an inert gas atmosphere.
9. The method according to claim 1, wherein the molecular distillation treatment in step (3) is carried out under conditions of a vacuum degree of 0.01 to 0.2pa, a transmembrane rotation speed of 250 to 350rpm, a feed rate of 1.0 to 1.5ml/min, and a distillation temperature of 120 to 160 ℃.
10. The method as claimed in claim 1, wherein the stirring rate of the hydrolysis reaction in step (1) is 200-1000 rpm; the stirring speed of the hydrolysis reaction in the step (2) is 200-1000 rpm.
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